The ability to detect bacterial contamination is paramount to improving food safety. During food processing, food can become contaminated with bacteria and spoil. Food poisoning can result if food contaminated with pathogenic bacteria, or its toxic products, is ingested without proper cooking.
Standard culture plate methods for monitoring surfaces for bacterial contamination require a sterile sample collection device (generally a swab or sponge) and suitable culture media, which after inoculation, must be incubated at a controlled temperature for a minimum of several hours to days. These methods are too cumbersome and time consuming, especially if used by untrained workers. Rapid bacteria tests need to be implemented in slaughterhouses and food handling establishments to improve safety. In these establishments, one must rapidly determine whether additional cleaning is required or whether proper safety procedures have been followed. To do that, a quick, reliable bacteria measurement is needed. Unfortunately, this is often not possible because present methods require several hours or even days by trained laboratory technicians or require elaborate testing equipments that are not readily transportable to on site locations.
Attempts have been made to overcome these shortcomings with more sensitive chemiluminescence detection methods. One such chemiluminescence method measures adenosine triphosphate (ATP) to indirectly measure the bacteria content. This detection is reliable because all bacteria contain some ATP. Chemical bond energy from ATP is utilized in the chemiluminescent reaction occurring, for example, in the tails of the firefly Photinus pyralis. The mechanism of this chemiluminescence reaction has been well characterized (DeLuca, M., et al, 1979 Anal. Biochem. 95:194-198). The components of this reaction can be isolated free of ATP and subsequently used to detect ATP in other sources by a reaction that begins with formation of an enzyme bound luciferyl-adenylate complex and free inorganic pyrophosphate and ends with a rapid reaction of this complex with molecular oxygen to produce light, CO.sub.2, and adenosine monophosphate (AMP).
One conventional light measuring method involves counting photons with a light measuring instrument. Photographic films also have been used to monitor chemiluminescent reactions, as described for example in U.S. Pat. No. 4,396,579. A drawback of these types are that they are complex and difficult to use.
Firefly luciferin-luciferase reactions have been used for detecting microorganisms, as described in U.S. Pat. Nos. 4,385,113 and 5,366,867. These methods, however, suffer a number of deficiencies. Lyophilized luciferase-luciferin reagent is unstable at room temperature during long term storage and is unstable after liquid reconstitution over short time intervals. Additionally, after reconstitution, the reagent solution emits significant amount of light even in the absence of ATP, which decreases detection sensitivity.
The reagent instability problem was partly addressed by drying luciferin-luciferase reagents separately onto plastic surfaces. But this required an additional step of transferring microorganisms from a collection device to a plastic surface, increasing complexity. Further, while this solves the instability problem, it unfortunately lowers the detection sensitivity and creates a new problem--incomplete ATP transfer from the collection device to a separate plastic surface containing the luciferase-luciferin reagent. Furthermore, this solution introduces a new time variable between the transfer and the light emission measurement.
Adding reagent at timed intervals causes additional problems because the light emission kinetics become shorter as the light intensity decreases. The twin timing and reagent instability problems also plague other so chemiluminescence chemistries that have been developed to detect target analytes. For example, U.S. Pat. No. 4,396,579 describes a complicated, expensive automated machine designed to add chemiluminescent reagent at fixed time intervals to overcome the light emission kinetic problem. The reagent instability and the timing problems make this machine unusually complex.
Thus, there is a need for an assay device that benefits from high sensitivity and speed of chemiluminescence detection, but one that excludes the aforementioned complexity, timing, reagent instability, and high background light emission problems. Copending U.S. patent application Ser. No. 08/560,094, filed Nov. 17, 1995 (hereafter "first copending application"), entitled CHEMILUMINESCENT ASSAY METHODS AND DEVICES FOR DETECTING TARGET ANALYTES, describes chemiluminescent assay methods and devices that fulfill this need. The disclosure of the first copending application, including its drawings, is incorporated herein by reference.
The first copending application discloses a simple, easy to use chemiluminescent sampling assay device that eliminates or reduces the complexity associated with manually measuring and adding reagent at timed intervals. This sampling device also provides means to measure light intensity and allows rapid analysis of target analytes at the sample site. Specifically, it comprises a container or envelope holding a sampling strip having separate sampling and reagent portions. The reagent portion contains one or more dried chemiluminescent reagents. The device has a light-permeable portion that permits light generated by a chemiluminescent reaction within the sampling strip to exit the container. This sampling assay device eliminates or reduces much of the complexity associated with other known assay methods and, as a result, decreases the cost and training requirements for detecting target analytes. A wide range of target analytes can be detected by this device. In fact, the sampling portion of the device can collect virtually any type of target analyte containing ATP, not only from physical contact with a solid, but also from sample liquid applied or introduced thereto. The advantage of rapid and sensitive detection of bacteria can be realized through sensitive light detection using, for example, a photomultiplier. The first copending application discloses, in essence, a compact, self-contained assay device that allows light detection using any of known light detection methods, including an optical observation.
Notwithstanding the advantages and benefits of the ATP detection method described in the first copending application, the ATP detection method tests for presence of microorganisms, not a specific microorganism, since all microorganisms contain ATP. In this regard, it would be desirable to detect specific microorganisms.
Conventional instruments for measuring chemiluminescence, including luminometers and fluorometers, however, are not particularly suited for such an assay device that has a flat geometry. To this end, there is a need for a portable interface readily interfaceable with a photomultiplier or other known light detector to provide a simple, efficient light intensity reading from the sampling assay device of the type disclosed in the first copending application. Copending U.S. patent application Ser. No. 08/577,107, filed Dec. 22, 1995 (hereafter "second copending application"), entitled SAMPLING-ASSAY INTERFACE SYSTEM AND METHOD describes a system that fulfills this need, the disclosure of which, including its drawings, is incorporated herein by reference.
Specifically, the second copending application describes a sampling-device holder interface system and a method for performing an assay for a target analyte from a sampling device of the type disclosed in the first copending application. The sampling system includes a sampling-device holder interface (hereafter "interface") and a quantifier for converting the output signal to quantifiable data indicative of the amount of the target analytes. Specifically, the interface comprises a sampling-device holder and a light detector--means for converting light generated from the sampling device to an output signal corresponding to the amount or intensity of the light generated--such as a photomultiplier or photodetector.
The interface holds a sampling device, which comprises a container and a sampling strip inside the container. The sampling strip has a sampling portion for introducing a sample, a reading portion containing a reagent for producing a chemiluminescent reaction with the target analytes, and a transfer portion connecting the sampling and reading portions for transferring the sample from the sampling portion to the reading portion. The container has an opening to permit introduction of samples to the sampling portion. It also has a light transmissive portion, such as a window or opening, visibly exposing the reading portion.
The holder includes a housing and a tray. The housing has at least first and second walls forming a cavity therebetween. One of the first and second walls has an opening or light transmissive window. The tray is received in the cavity and movable between opened and closed positions. The tray has a compartment adapted to seat and support the sampling assay device. The first opening is in registry with the reading portion when the tray is in the closed position to enable observation of the reading portion through the first opening. The light detector is connected to the housing, in registry with the first opening. The tray has a second opening extending through the compartment, which opening is in registry with the reading portion of the seated sampling device. When the tray is in the closed position, the second opening is in registry with the first opening to enable observation of the reading portion through both the first and second openings.
Not withstanding the advantages and benefits derived from the sampling device and the interface system adapted for the sampling device disclosed in the first and second copending applications, the interface system is not adapted for the luminescent (fluorescent or phosphorescent) light detection. Another potential drawback with this type of sampling device is that the chemiluminescent reaction takes place spontaneously as the chemiluminescent reagent mixes with the target analyte. In this regard, it is not possible to selectively take measurements or delay measurements once sampling is initiated. Hence, there is a need to selectively trigger luminescent reaction independently of sampling. Copending U.S. patent application Ser. No. 08/580,096, filed Dec. 22, 1995 (hereafter "third copending application"), entitled SAMPLING-ASSAY DEVICE, INTERFACE SYSTEM, AND METHOD, describes a luminescent (fluorescent or phosphorescent) assay method and device that fulfill this need. The disclosure of the third copending application, including its drawings, is incorporated herein by reference.
Specifically, the third copending application describes a sampling device, an interface for holding the sampling device, and a system and method thereof for performing an assay for a target analyte from a sample using luminescent light detection. The sampling device comprises a sampling strip housed in a container. The sampling strip has a sampling portion for receiving a sample, a reading portion for emitting light, and a transfer portion connecting the sampling and reading portions for permitting transfer of the sample from the sampling portion to the reading portion. The reading portion contains an immobilized binding agent complementary to the target analyte. This enables the reading portion to capture or immobilize the target analyte within the reading portion while allowing non-captured elements to pass through or exit the reading portion. Specifically, the binding agent preferably is an antigen complementary to the target analyte.
One or both the sampling portion and the transfer strip contains a luminescent (fluorescent or phosphorescent) labeling agent, which glows when exposed to light. The labeling agent, which preferably is chelated europium or europium compound, is contained within at least a portion of the transfer portion near or adjacent the reading portion. The labeling agent is bound to another binding agent complementary to the target analyte. Thus, the labeling agent specifically binds to the target analyte.
The container comprises a first layer and a second layer sandwiching the sampling strip and has means, which preferably is a first opening formed through the first layer and aligned with the sampling portion, to permit introduction of the sample to the sampling portion, and has a light transmissive portion exposing the reading portion. The light transmissive layer is preferably a second opening though the second layer and aligned with the reading portion. The second layer includes a light transmissive member to cover at least the second opening.
The interface comprises a sample holder, a light detector for converting light emitted from the sampling device to an output signal corresponding to the amount or intensity of the light generated, such as a photodetector or photomultiplier, connected to the holder and a light source, such as an LED, laser diode, or gas-filled lamp, connected to the holder. The holder comprises a housing having a first wall and a second wall. The first and second walls form a cavity therebetween, with the first wall having a first opening. A tray is received in the cavity and movable between an opened position and a closed position. The tray also has a compartment adapted to receive and support the sampling device and a second opening extending through the compartment. The second opening is in registry with the reading portion when the sampling device is seated in the compartment and in registry with the first openings to enable observation of the reading portion through the first and second openings.
The light detector has a light gathering window and is connected to the first wall so that the window is aligned with the first opening. The light source is aligned with the first opening and connected opposite the first opening. Specifically, the second wall has a third opening aligned with the first opening and the light source is seated in the third opening. The second wall is an enclosure having a channel and the first wall is a base plate connected to the enclosure, the channel defining the cavity.
When the tray is in the opened position, the tray blocks the first opening. The tray can also include a handle and is preferably slidable between the opened and closed positions. In this regard, the tray includes a pair of parallel slots, which are adapted to be occupied by fasteners spaced along the slots. The length of the slot less the spacing between the fasteners occupying the same slot defines the amount of the tray sliding movement. The system is adapted for use with the aforedefined sampling device and includes the aforedefined interface, and further includes a quantifier, such as an ammeter, for converting the output signal-from the light detector to quantifiable data indicative of the amount of the target analyte.
The sampling device disclosed in the first, second, and third copending applications provides a unique means for allowing light detection using known light detection devices. Because the sampling device, however, is, rather flexible, thin, and flat, it can be challenging to remove the same from the tray compartment. The sampling device needs to be removed by prying out with a fingernail or some sharp instrument. One can also turn the interface upside down and drop the sampling device. But in any event, it would be desirable to ease the sampling device removal from the tray. In addition, there is a need to protect the exposed sampling portion from cross contamination. For instance, if the interface or holder is shaken or otherwise turned sideways or upside down, it is possible for the exposed sampling portion to contact the underside of the housing upper wall, which contact could possibly introduce other samples that made contact therewith. Copending U.S. patent application Ser. No. 08/577,624, filed Dec. 22, 1995 (hereafter "fourth copending application"), entitled SAMPLING-ASSAY DEVICE, describes a sampling assay device for use with chemiluminescent and luminescent (fluorescent or phosphorescent) that fulfills this need. The disclosure of the fourth copending application, including its drawings, is incorporated herein by reference.
The fourth copending application describes a sampling device having a container and a sampling strip inside the container. The sampling strip has a sampling portion for receiving a sample, a reading portion for holding the sample with a compound that can emit light, and a transfer portion connecting the sampling and reading portions for permitting transfer of the sample from the sampling portion to the reading portion. The container has means to permit introduction of the sample to the sampling portion and a light transmissive portion exposing the reading portion. A shield extends from the container adjacent the exposed sampling portion. The shield has a portion extending beyond one end of the container and is movable to and from the sampling portion and can be wrapped around the one end.
Specifically, the container comprises a first layer and a second layer sandwiching the sampling strip. The shield is attached to or integral with the first layer and has means for permitting the extending portion to wrap around the one end of the container. The wrap around means is preferably a preformed fold or crease, or even a perforation. A tab or handle is attached to or formed integrally with the first layer. Alternatively, the tab can also be attached to or integral with the shield.
The sample introducing means preferably is a first opening formed through the first layer and aligned with the sampling portion. Similarly, the light transmissive layer is preferably a second opening though the second layer and aligned with the reading portion. The second layer preferably also includes a light transmissive member to cover at least the second opening.
According to one embodiment, the sampling strip is composed of an adsorbent material. In another embodiment, the sampling strip includes a poly-carbonate membrane that is light transmissive.
The sampling device can further include a sample collecting member, which preferably is adsorbent, in contact with the sampling portion inside the container and aligned with the first opening. The sample collecting member receives the sample and transfers the sample to the sampling portion.
According to one embodiment, the compound is a reagent, preferably an enzyme in a dried state, contained within the reading portion. When the reagent is mixed with the analyte, it produces chemiluminescent light. More preferably, the reagent is luciferase-luciferin in a dried state.
According to another embodiment, the compound, which preferably is a luminescent labeling agent (phosphorescent or fluorescent) with a binding agent for tagging the analyte, is contained within at least a portion of the transfer portion. The labeling agent glows when it is exposed to light. Preferably, the labeling agent is chelated europium or europium compound. The reading portion contains an immobilized binding agent, such as an antigen, specific to the analyte, for capturing the labeled analyte within the reading portion. In this embodiment, the sampling strip further includes a collecting portion contiguous with the reading portion. This collection portion is designed to absorbs any excess liquid containing the labeling compound not coupled to the binding agent. Accordingly, as the analyte immobilized in the reading portion carries the labeling agent that glows when exposed to light, the amount of light produced after exposure to light correlates to the amount of analyte present in the sample. In operation, as the sample contained in liquid travels across the transfer portion, the target analyte will pickup the labeling agent. Other organisms mixed with the labeling compound and the excess labeling compound, however, are not specific to the binding agent. Thus, they will not be captured in the reading portion, but rather will flow through. The target analyte, however, since it is specific to the binding agent, will be captured and remain in the reading portion.
The sampling device according to the fourth copending application is particularly adapted for use with a sampling-device holding interface, which has a housing and a tray for seating the sampling device, as described in the second and third copending applications. The housing has a cavity for accepting the tray with the sampling device. The cavity is light-light tight when the tray is closed. The housing is connected to a light detector or the like to measure the amount of light generated by the sample. The housing can have also have a light source for triggering reaction of the luminescent labeling agent.